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目的:EB病毒(Epstein-Barr virus,EBV)感染宿主细胞包括潜伏期和裂解期,它的即刻早期基因BRLF1编码产物Rta蛋白是EBV从潜伏期到裂解期的关键性调节因子。目前,Rta蛋白已成为治疗EBV相关肿瘤的靶蛋白,但有关BRLF1基因在EBV阳性淋巴瘤中的研究甚少,本研究旨在明确EB病毒即刻早期基因BRLF1在EB病毒阳性淋巴瘤组织中的变异规律,探讨其变异意义。方法:原位杂交筛选EB病毒阳性淋巴瘤(BL),提取EB病毒DNA。PCR结合DNA测序检测EBV阳性淋巴瘤中BRLF1基因多态性,应用DNAStar软件对序列进行对比分析。结果:共筛选出27例EBV阳性淋巴瘤标本,在27例BL阳性标本中成功扩增BRLF1基因,与B95-8标准序列比对分析,共发现20处无义突变和19处有义突变。8例属于BR1-A亚型,18例属于BR1-C亚型,1例属于BR1-E亚型,其中以BR1-A亚型和BR1-C亚型最为常见,分别为29.6%(8/27)、66.7%(18/27)。与EBVaGC、NPC和TW相比,4种人群BRLF1亚型的分布不同,其中BR1-A亚型更多出现在健康人群中,BR1-C亚型更多出现在NPC和BL中。在Rta蛋白功能区中,二聚体区域呈高度保守,在DNA结合区域和反式激活区域均检测出序列突变,16种CTL抗原表位中,只有NAA、QKE和ERP表位发生改变,且NAA和QKE在健康人群中突变率较高。结论:山东地区EB病毒阳性淋巴瘤BRLF1基因变异类型主要为BR1-A亚型和BR1-C亚型。BR1-C亚型可能与BL相关,发生在Rta蛋白DNA结合区域和反式激活区域的突变可能对其功能产生影响,多数CTL表位序列保守提示BRLF1可以用作EBV相关淋巴瘤免疫治疗的靶基因。
OBJECTIVE: Epstein-Barr virus (EBV) infects host cells including the latent period and the lytic phase, and its immediate early gene BRLF1-encoding product Rta protein is a key regulator of EBV from latency to lysis. At present, Rta protein has become the target protein for the treatment of EBV-related tumors. However, little is known about the BRLF1 gene in EBV-positive lymphomas. The aim of this study was to determine the mutation of BRLF1 in EBV-positive lymphoma tissue Law, explore the significance of its variation. Methods: Epstein - Barr virus positive lymphoma (BL) was screened by in situ hybridization and Epstein - Barr virus DNA was extracted. PCR was used to detect the polymorphism of BRLF1 gene in EBV positive lymphoma by DNA sequencing. DNAStar software was used to compare the sequences. Results: A total of 27 specimens of EBV positive lymphoma were screened and BRLF1 gene was amplified successfully from 27 BL positive specimens. Comparing with B95-8 standard sequence, 20 nonsense mutations and 19 sense mutations were found. 8 cases belonged to BR1-A subtype, 18 cases belonged to BR1-C subtype and 1 case belonged to BR1-E subtype, of which BR1-A subtype and BR1-C subtype were the most common, which were 29.6% 27), 66.7% (18/27). Compared with EBVaGC, NPC and TW, the distribution of BRLF1 subtypes was different in four populations, with BR1-A subtype appearing more in healthy people and BR1-C subtype more in NPC and BL. In the Rta protein functional region, the dimer region was highly conserved, with sequence mutations detected in both the DNA binding and transactivation regions, of which only the NAA, QKE and ERP epitopes were altered in the 16 CTL epitopes and The mutation rate of NAA and QKE is higher in healthy people. Conclusion: The variants of BRLF1 gene in Epstein-Barr virus positive lymphoma in Shandong Province are mainly BR1-A subtypes and BR1-C subtypes. BR1-C subtypes may be associated with BL, and mutations in the DNA-binding and transactivation regions of Rta may have an impact on their function. Most CTL epitope sequences suggest conservatively that BRLF1 can be used as a target for EBV-related immunotherapy gene.